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Department of Energy,Joint Genome Institute, Walnut Creek, CA 94598, USA; 2Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; 3Plant Gene
Expression Center, USDA-ARS, Albany, CA 94710, USA; 4Departamento de Ingeniera Genetica, Centro de Investigacion y de Estudios Avanzados, Irapuato 36821, Mexico; 5Department of
Biology, Occidental College, Los Angeles, CA 90041, USA; 6School of Natural Sciences, University of California, Merced, CA 95343, USA
Summary
Authors for correspondence:
Axel Visel
Tel: +1 510 495 2301
Email: avisel@lbl.gov
Laila P. Partida-Martinez
Tel: +52 462 623 9658
Email: laila.partida@ira.cinvestav.mx
Susannah G. Tringe
Tel: +1 925 296 5813
Email: sgtringe@lbl.gov
Received: 1 July 2015
Accepted: 31 August 2015
Desert plants are hypothesized to survive the environmental stress inherent to these regions
in part thanks to symbioses with microorganisms, and yet these microbial species, the communities they form, and the forces that influence them are poorly understood.
Here we report the first comprehensive investigation of the microbial communities associated with species of Agave, which are native to semiarid and arid regions of Central and North
America and are emerging as biofuel feedstocks. We examined prokaryotic and fungal communities in the rhizosphere, phyllosphere, leaf and root endosphere, as well as proximal and
distal soil samples from cultivated and native agaves, through Illumina amplicon sequencing.
Phylogenetic profiling revealed that the composition of prokaryotic communities was
primarily determined by the plant compartment, whereas the composition of fungal communities was mainly influenced by the biogeography of the host species. Cultivated A. tequilana
exhibited lower levels of prokaryotic diversity compared with native agaves, although no
differences in microbial diversity were found in the endosphere.
Agaves shared core prokaryotic and fungal taxa known to promote plant growth and
confer tolerance to abiotic stress, which suggests common principles underpinning Agave
microbe interactions.
Key words: Agave, biogeography, cultivation, desert, iTags, microbial diversity, plant
microbiome, plantmicrobe interactions.
Introduction
The genus Agave, native to the deserts and dry lands of central
Mexico and the southwestern USA, includes a number of
promising candidate biofuel crops for arid and semiarid climates (Somerville et al., 2010; Davis et al., 2011). There are
> 200 species of Agave, and several of these have been cultivated for centuries for the production of alcohols and fiber
(Garcia-Moya et al., 2011). Recent research has highlighted the
suitability of agaves as biofuel feedstocks as a consequence of
their comparatively low lignin content, ease of decomposition,
and high yields with minimal resource inputs (Davis et al.,
This manuscript has been authored by an author at Lawrence Berkeley
National Laboratory under Contract no. DE-AC02-05CH11231 with the
US Department of Energy. The US Government retains, and the publisher,
by accepting the article for publication, acknowledges, that the US
Government retains a non-exclusive, paid-up, irrevocable, world-wide license
to publish or reproduce the published form of this manuscript, or allow
others to do so, for US Government purposes.
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Gentry et al., 2013), their differentiated management status (cultivated versus native), and their relevance to both existing and
prospective industries. Comparisons across these three Agave
species would allow us to infer the impact of biogeography and
cultivation status on microbial composition and diversity in the
genus Agave. Additionally, we also evaluate the major biotic (i.e.
plant species and plant compartment) and abiotic (i.e. season and
site) factors shaping both prokaryotic and fungal communities,
and identify prominent microbial players in order to develop
rational microbiome-based strategies for improving the yield and
productivity of Agave.
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(a)
(b)
(c)
(d)
(e)
the ITS2 and 16S regions, we used the ITS9F/ITS4R and 515F/
816R primer pairs, respectively, and followed the PCR protocol
described in the Supporting Information. For 16S amplification,
peptide nucleic acid (PNA) clamps were used as in Lundberg
et al. (2013), which substantially reduced overall chloroplast and
mitochondrial contamination and increased the proportion of
prokaryotic reads for the root endosphere, leaf endosphere, and
phyllosphere samples. Paired-end 2 9 250 bp sequencing was
performed on an Illumina MiSeq instrument (Illumina Inc., San
Diego, CA, USA) operating with v2 chemistry. All quality
sequences related to this project are available in the NCBI
Sequence Read Archive (SRA) under project IDs SRA211411,
SRA211420, SRA211416, SRA211422 and SRA211408.
Data processing and statistical analyses
The raw Fastq reads were processed using a custom pipeline
developed at the Joint Genome Institute (Tremblay et al., 2015)
(Supporting Information Methods S1). Raw reads were contaminant-filtered, quality trimmed, merged and clustered to produce 25 871 and 40 759 fungal and prokaryotic operational
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taxonomic units (OTUs), respectively, at 95% and 97% identity using the UPARSE pipeline (Edgar, 2013). Taxonomies
were assigned to each OTU using the RDP Nave Bayesian
Classifier (Wang et al., 2007) with custom reference databases.
OTUs whose RDP classifications did not match their expected
taxonomic kingdoms (Fungi and Bacteria/Archaea, respectively)
were removed. Average read counts varied by sample type for
both data sets, and in particular the aerial endophytic samples
had substantially fewer reads than the other sample types
(Fig. S1). To reduce low-abundance and spurious OTUs, technical reproducibility thresholds determined empirically from
technical replicates as in Lundberg et al. (2013) (Fig. S2) were
set and OTUs kept only if they had at least two reads in at least
five samples (ITS2 data) or at least seven reads in at least five
samples (16S data).
For diversity analyses, all samples were randomly subsampled
(rarefied) to 1000 reads per sample to account for differences in
the number of reads across samples. We calculated the Shannon
diversity (H0 ) index using the package BiodiversityR in R. All
other statistical analyses were performed in R using a variety of
packages and custom scripts using the measurable OTUs, as
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Table 1 Geographic location, annual mean temperature, precipitation and soil characteristics of study sites
Sites
Latitude
Longitude
Altitude (m above sea level)
Annual mean temperature (C)a
Annual precipitation (mm)a
Precipitation during dry season (mm)a
Precipitation during rainy season (mm)a
Soil edaphic factors
Texture
pHb
Organic matter (%)
Nitrogen (lg g1)
Phosphorus (lg g1)
Potassium (lg g1)
Mexico
USA
Guanajuato
Jalisco
Agave tequilana
California
A. salmiana
Amatitan
(Am)
Penjamo
(Pe)
El Magueyal
(Ma)
San
Felipe (SF)
Boyd
Ridge (BR)
Agave
Hill (AH)
Pinyon
Flats (PF)
21.053
103.902
1260
26.4
558
4.2
553.8
Clay loam
6.8
1.1
19.7
3.64
193
20.686
101.875
1714
19
790
147
643
Clay
6.68
0.96
9.14
27.1
482
21.195
100.439
2175
17.9
485
130
355
Sandy loam
5.79
3.97
8.76
18.11
64.35
21.766
100.163
2089
17
204.5
68
136.5
Sandy loam
6.25
0.71
12.65
4.53
251.7
33.714
116.524
452
23.7
136.0
51.7
83.9
Sandy loam
8.02
nd
nd
42.17
155.5
33.665
116.425
814
20.6
183.0
69.4
113.6
Sandy loam
7.48
nd
nd
87
131
33.601
116.595
1225
18.0
238.3
95.5
142.8
Sandy loam
7.95
nd
nd
74
91.33
A. deserti
a
n Nacional del Agua (CONAGUA). Data from sites in California were obtained from DRI Weather
For sites in Mexico, data were provided by Comisio
Station Data Collection (http://www.wrcc.dri.edu/weather/ucde.html). nd, not determined.
b
Statistically significant difference between species after KruskalWallis test (H2,15 = 10.763; P = 0.0046).
greater statistical support was achieved using this data set instead
of the measurable rarefied data set. In brief, Venn diagrams were
plotted with the function venn.diagram using the package
VennDiagram. Distances were calculated using the vegdist
function of the package vegan for BrayCurtis. Nonmetric multidimensional scaling (NMDS) was performed using Mass and
vegan packages. Permutational ANOVAs (PERMANOVAs)
were performed with the function adonis in the package vegan
as described in Desgarennes et al. (2014). The major microbial
player analyses and graphics were performed using the
average relative abundance and relative frequency of each
OTU in each community (i.e. sample) across the three Agave
species.
Results
Microbial community diversity associated with Agave
species
We analyzed the prokaryotic and fungal communities associated with six sample types taken from three Agave species
through Illumina iTag sequencing on the MiSeq platform.
We obtained 35 770 987 and 27 596 665 total high-quality
reads (Fig. S1), which resulted in 3923 and 3173 OTUs after
applying technical reproducibility thresholds for the prokaryotic and fungal data sets (Fig. S2), respectively. The majority
of all prokaryotic and fungal OTUs discovered in the endospheres (leaf and root) in this analysis were also present in
the episphere (rhizosphere and phyllosphere) and surrounding
soils (Fig. 1d). Similarly, the majority of OTUs associated
with the aboveground portions of the plant were also present
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(a)
(b)
high as diversity in the rhizosphere, despite the harsher environmental conditions that prevail for aboveground plant surfaces
than for underground surfaces.
Across all samples, we detected a total of 33 distinct prokaryotic phyla, although just four of these (Proteobacteria,
Actinobacteria, Firmicutes and Bacteroidetes) comprised on average
> 80% of each plant-associated sample type (rhizosphere, root
endosphere, leaf endosphere, and phyllosphere; Fig. S4). The
plant-associated sample types (epispheres and endospheres) were
enriched for Proteobacteria (KruskalWallis v2 = 50.84;
P < 1.00 9 1012)
and
Actinobacteria
(KruskalWallis
v2 = 11.07; P < 8.790 9 104) and depleted for Acidobacteria
(KruskalWallis v2 = 125.51; P < 2.20 9 1016) with respect to
the surrounding soils, consistent with previous observations for
Agave and other plants (Bulgarelli et al., 2012; Lundberg et al.,
2012; Shakya et al., 2013; Desgarennes et al., 2014). While the
prokaryotic relative abundance profile seemed to be similar in the
bulk and root zone soil samples, community composition differed substantially between the rhizosphere, root endosphere, leaf
endosphere, and phyllosphere (Fig. 3). By contrast, the transition
in relative abundance profiles between sample types for fungal
communities was more gradual than discrete (Fig. 3), with several
lineages exhibiting proportional increases or decreases in abundance across bulk soil, root zone, rhizosphere and root endosphere compartments. Fungal communities across all six sample
types were dominated by the phylum Ascomycota (average 91.2%
of total relative abundance), while Basidiomycota represented a
much smaller portion of the communities (7.7%) (Fig. S4).
Strikingly, very few members of the known arbuscular
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(a)
(b)
(c)
(d)
(e)
(f)
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Table 2 PERMANOVA analyses of the microbial communities associated with Agave species considering all factors and their interactions (only significant
factors are displayed; P 0.05)
Prokaryotes
Factora
R2
Global
Sample type5,199
Species2,199
Sample type: species10,199
183.247
107.096
10.517
0.631
0.148
0.072
88.056
Fungi
Factora
R2
0.001
0.001
0.001
Species2,190
Sample type5,190
Sample type: species10,190
Sample type: season5,190
Season1,190
350.97
49.04
16.83
1.94
2.54
0.527
0.184
0.126
0.007
0.002
0.001
0.001
0.001
0.016
0.050
0.667
0.001
Species2,71
Sample type1,71
190.848
4.284
0.820
0.009
0.001
0.019
88.016
32.056
7.142
0.591
0.108
0.048
0.001
0.001
0.001
Species2,70
Sample type: species2,70
Sample type1,70
Season1,70
254.033
34.259
56.940
2.985
0.709
0.096
0.080
0.004
0.001
0.001
0.001
0.049
110.965
12.546
7.274
5.228
4.624
3.364
0.479
0.108
0.063
0.045
0.020
0.014
0.001
0.001
0.001
0.001
0.014
0.027
Sample type1,49
Species2,49
Sample type: species2,49
Season1,49
Sample type: season1,49
Sample type: species: season1,49
57.269
17.771
4.451
3.756
3.513
2.617
0.351
0.218
0.055
0.023
0.022
0.016
0.001
0.001
0.001
0.008
0.014
0.049
Subscript numbers indicate the degrees of freedom and residuals of each F test.
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0.4
(b)
Coordinate 2
0.4 0.2 0.0 0.2 0.4
0.0
-0.2
0.6 0.4
Coordinate 2
0.2
(a)
0.6
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0.5
0.0
0.5
1.0
0.4
0.2
0.0
Coordinate 1
0.2
0.0
0.5
0.0
Coordinate 2
0.5
0.4
0.2
0.4 0.2 0.0
Coordinate 2
0.4
(d)
0.6
(c)
1.0
0.6
0.4
0.2
Coordinate 1
0.0
0.2
0.4
Coordinate 1
Coordinate 2
0.5
0.0
(f)
0.6 0.4 0.2 0.0 0.2 0.4 0.6
(e)
Coordinate 2
0.2
Coordinate 1
0.6
0.5
0.4
Coordinate 1
Species
Bulk Soi l
0.2
0.0
0.2
0.4
0.6
Coordinate 1
Root Zon e
Rhizosphere
Root
Leaf
Endosphere
Endosphere
Phyllosphere
A. tequilana
A. salmiana
A. deserti
Fig. 4 Nonmetric multidimensional scaling (NMDS) plots for BrayCurtis distances of prokaryotic and fungal communities associated with Agave species.
(a, b) All six sample types; (c, d) only rhizospheres and phyllospheres; (e, f) only root and leaf endospheres.
the factors site and season did influence both prokaryotic and
fungal communities (Table S1), suggesting that abiotic factors
played a more substantial role in shaping the microbiome of
A. tequilana.
Major microbial players associated with Agave
the endosphere
Inspired by the possibility of approaching ecological relationships
as microbial markets (Werner et al., 2012), we made use of the
Pareto concept (the 8020 rule) to identify those prokaryotic and
fungal taxa accounting for 80% of Agaves microbial communities. After their identification, we plotted their average relative
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(a)
(b)
Fig. 5 Relative frequency versus relative abundance of major operational taxonomic unit (OTU) players associated with the root endosphere of agaves for
(a) prokaryotes and (b) fungi. Arrows indicate most abundant genera in each case. The number of OTUs and the number of samples (n) are indicated in
the bottom right corner of each plot.
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(a)
As.Ma As.Sf At.Am At.Pe
Dry
Leaf
endosphere
Root
endosphere
Rainy
Leaf
endosphere
Root
endosphere
(b)
Bacterial Taxa
Crenarchaeota
Acidobacteria
Actinobacteria
Flavobacteria
Sphingobacteria
Chloroflexi
Bacilli
Clostridia
Gemmatimonadetes
Nitrospirae
Planctomycetes
Alphaproteobacteria
Betaproteobacteria
Deltaproteobacteria
Gammaproteobacteria
Verrucomicrobia
S14; Tables S4, S5). This may be explained by the fact that
A. deserti occupies the driest habitat of the three species examined
(Nobel, 2003), experiencing smaller seasonal variations.
In contrast to these differences observed for prokaryotic communities, fungal endophytic communities were less affected by
season (Fig. S15; Table S5). At higher taxonomic levels, the leaf
endospheres associated with A. tequilana and A. salmiana were
similar to one another across both dry and rainy seasons
(Fig. S15). In general, while fungal root endosphere communities
of the three Agave species could be differentiated from one
another (Fig. 4f) based on total relative abundance patterns, they
did in fact share a core of eighty OTUs representing c. 40% or
more of the total community in each species (Figs S14, S15).
Discussion
Distinct factors shape prokaryotic and fungal communities
in Agave
The microbial communities associated with plant hosts are probably shaped by a wide variety of environmental and host-related
factors, including geographic location, plant phenotype and
genotype, soil chemistry, and seasonal effects. This study determined that prokaryotic communities associated with agaves were
chiefly influenced by the sample type or plant compartment,
where the rhizosphere, the phyllosphere and the root and leaf
endosphere were clearly distinct from one another and also from
the surrounding soils. These results were previously suggested for
agaves (Desgarennes et al., 2014), but by using next-generation
sequencing (NGS), these differences were made more clear even
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investigated by comparing microbial communities of agaves with
those associated with other sympatric Agave or non-Agave species
in these arid environments. Recent studies have demonstrated
that plant host-specific traits, including broad morphological
characteristics (Kembel et al., 2011) and specific genetic pathways
and gene products (Horton et al., 2014; Lebeis et al., 2015), can
have significant effects on microbiome composition and diversity.
In the endosphere, an influence of the season was observed in the
Mexican agaves (Figs 6, S13; Table S4), especially in the prokaryotic communities, as detected previously (Desgarennes et al.,
2014). Leaf endophytic fungal communities were similar in
A. tequilana and A. salmiana, which suggests common mechanisms of plantfungus interaction in this habitat.
Low levels of microbial diversity in the cultivated
A. tequilana compared with native agaves
Cultivated agaves are susceptible to a number of diseases, many
of which cause significant losses in yield and revenue each year
(Dalton, 2005). One of the most costly of these diseases, called
soft rot, was previously estimated to have cost the tequila industry $200 million yr1 (Jimenez-Hidalgo et al., 2004). A putative
causative agent of soft rot has been identified as a member of
the bacterial family Enterobacteriaceae (Pantoea agglomerans)
(Jimenez-Hidalgo et al., 2004). In this study, we observed significantly lower levels of prokaryotic diversity in the rhizosphere and
phyllosphere of the cultivated A. tequilana compared with the
native A. salmiana and A. deserti. Furthermore, this lower alpha
diversity was the direct result of the dominance in the epispheres
of A. tequilana of a few bacterial families, including genera
belonging to Enterobacteriaceae (Pantoea, Leclercia, and
Trabusiella). The lack of enrichment for Enterobacteriaceae in the
surrounding bulk soils suggests that these bacterial species have
adapted to specific associations with their plant host. A number
of agronomic and regulatory practices within the tequila industry
may be potentially influencing prokaryotic alpha diversity in
A. tequilana. First, continuous monocultures, as is typically the
case on agave plantations, have been shown to decrease microbial
community diversity (Hilton et al., 2013). Second, tequila plantations typically grow young agave plants indoors for a period of
time before transplanting to the field, and, before transplant, the
roots are sterilized in formaldehyde and left to dry for a period of
time (Davis et al., 2011). As rhizosphere microbial communities
have been shown to assemble very early in plant development
(Chaparro et al., 2014), root sterilization probably alters the progression and development of a natural microbial community.
Third, governmental regulations that limit the available genetic
pool and propagation techniques (vegetative reproduction via
bulbils and rhizome offshoots preferred) result in agave plantlets
that are genetically identical to the parental line (Valenzuela,
2011). High genetic homogeneity of the A. tequilana cultivar
used in the tequila industry may have allowed the pathogenic
microbes to evolve strategies to evade the limited arsenal of available host defenses. Current research aimed at adapting agave as a
biofuels feedstock is exploring the use of a variety of Agave
species, including the main cultivars from both the fiber and
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Acknowledgements
We thank Kanwar Singh (Joint Genome Institute) for technical
assistance during library construction, Derek Lundberg (Department of Biology, UNC) for support with the PNA protocol for
16S amplification, Edward Kirton (Joint Genome Institute) for
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Author contributions
S.G., D.C-D., G.N., T.W., A.V., L.P.P-M. and S.G.T. planned
and designed research; D.C-D., D.D., C.F-G. and S.G. performed experiments; S.G., G.N., D.D., C.F-G. and L.P.P-M.
conducted fieldwork; D.C-D. and S.C. prepared libraries and
processed sequencing data; D.C-D., D.D., C.F-G. and L.P.P-M.
analyzed the data; and D.C-D., D.D., A.V., L.P.P-M. and
S.G.T. wrote the manuscript. All authors read and approved the
final manuscript.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 Raw read counts across all samples grouped by sample
type.
Fig. S2 Established cut-off thresholds for technical reproducibility across OTUs for 16S and ITS2 data sets.
Fig. S3 OTU distributions across sample subsets.
Fig. S4 Phylum-level relative abundance plots of prokaryotic and
fungal communities associated with each Agave species by sample
type.
Fig. S5 Order-level relative abundance plots of prokaryotic communities associated with all rhizosphere and bulk soil samples.
Fig. S6 NMDS clustering for prokaryotic data of all samples
using unweighted and weighted UniFrac distances.
New
Phytologist
Fig. S8 Major prokaryotic taxa associated with Agave tequilana
based on the 8020 rule (Pareto).
Fig. S9 Major prokaryotic taxa associated with Agave salmiana
based on the 8020 rule (Pareto).
Fig. S10 Major prokaryotic taxa associated with Agave deserti
based on the 8020 rule (Pareto).
Fig. S11 Major fungal taxa associated with Agave tequilana based
on the 8020 rule (Pareto).
Fig. S12 Major fungal taxa associated with Agave salmiana based
on the 8020 rule (Pareto).
Fig. S13 Major fungal taxa associated with Agave deserti based on
the 8020 rule (Pareto).
Fig. S14 OTU distributions across Agave species in the endosphere.
Fig. S15 Fungal taxa associated with the endosphere of all three
Agave species and their relative abundance at the dry and rainy
seasons.
Table S1 Estimated Shannon diversity in the prokaryotic and
fungal communities associated with each Agave species
Table S2 PERMANOVA of the microbial communities associated with Agave tequilana considering all factors and their interactions
Table S3 PERMANOVA of the microbial communities associated with Agave salmiana considering all factors and their interactions
Table S4 PERMANOVA of the microbial communities associated with Agave deserti considering all factors and their interactions
Table S5 PERMANOVA of the microbial communities associated with the endosphere of Agave tequilana and A. salmiana considering all factors and their interactions
Methods S1 Methods for sample collection, DNA extraction,
PCR amplification, sequencing, and data processing, including
statistical analyses.
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